Method of inhibiting tumor necrosis factor

ABSTRACT

The invention provides a method of inhibiting TNF comprising administering a TNF-inhibiting amount of an extract of  Eleutherococcus senticosus maxim  to a subject. Also, the present invention provides a method inhibiting TNF in a cell comprising administering to the cell a TNF inhibiting amount of an extract from  Eleutherococcus senticosus maxim . Finally, the invention provides a method of treating a TNF mediated disorder in a subject, comprising administering a therapeutic amount of an extract of  Eleutherococcus senticosus maxim  to the subject.

[0001] This invention is a continuation-in-part of U.S. Ser. No. 07/953,051, filed Sep. 30, 1992, U.S. Ser. No. 07/953,052, filed Sep. 30, 1992, and U.S. Ser. No. 08/063,218, filed May 17, 1993, the contents of each of which are hereby incorporated by this reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to the use of Eleutherococcus senticosus maxim extract to inhibit tumor necrosis factor (TNF) in subjects including humans and cells and to treat disorders mediated by TNF.

[0004] 2. Background Art

[0005] It is becoming increasingly evident that tumor necrosis factor (TNF) is a cytokine involved in numerous pathologies afflicting subjects including humans and other animals. For instance, introduction of the endotoxin from gram(-) bacteria induces the production of cytokines including TNF. Excessive production of such cytokines is thought to be the cause of endotoxic or septic shock. In addition, TNF has been shown to activate latent HIV-infected cells in vitro and in vivo. (See Zinnser, Microbiology, 20th Ed. Appleton & Lange, 1992, at p. 1052.)

[0006] A crude extract derived from the root of Eleutherococcus senticosus maxim (E. maxim) has been utilized to treat various disorders. This Eleutherococcus senticosus maxim root extract is marketed under the name CM-4. In addition, Eleutherococcus sentidosus maxim extract has been marketed in combination with a spleen extract as PCM-4.

[0007]Eleutherococcus senticosus maxim extract has been used treat anemia, depression, diseases of the heart and blood vessels, the rigors of surgery, convalescence, tuberculosis, and aging. PCM-4 has also been used to treat HIV⁺ patients. However, despite the many therapeutic benefits associated with Eleutherococcus senticosus maxim extract, additional properties of Eleutherococcus senticosus maxim extract are continually being found which are of great value to mankind. The present application provides the surprising discovery and use of Eleutherococcus senticosus maxim extract as an inhibitor of TNF.

SUMMARY OF THE INVENTION

[0008] The present invention provides a method of inhibiting tumor necrosis factor (TNF) in a subject comprising administering to the subject a TNF inhibiting amount of an extract from Eleutherococcus senticosus maxim. Suitable subjects include humans. The Eleutherococcus senticosus maxim extract inhibits both TNF-α and TNF-β.

[0009] In addition, the present invention provides a method inhibiting TNF in a cell comprising administering to the cell a TNF inhibiting amount of an extract from Eleutherococcus senticosus maxim. Suitable subjects include humans. The Eleutherococcus senticosus maxim extract inhibits both TNF-α and TNF-β.

[0010] Finally, the present invention provides a method of treating a TNF mediated disorder in a subject, comprising administering a therapeutic amount of an extract of Eleutherococcus senticosus maxim to the subject. Suitable subjects include humans. The Eleutherococcus senticosus maxim extract treats disorders mediated by both TNF-α and TNF-β.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows the effect of Eleutherococcus senticosus maxim extract on TNF production in the HL-60 cell line.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention provides a method of inhibiting tumor necrosis factor (TNF) in a subject and in a cell comprising administering to the subject a TNF inhibiting amount of an extract from Eleutherococcus senticosus maxim. The method of the invention may be practiced upon subjects including humans. The Eleutherococcus senticosus maxim extract of the invention inhibits both TNF-α and TNF-β. In a further preferred embodiment the extract is CM-4.

[0013] Furthermore, the present invention provides a method of treating a TNF mediated disorder in a subject, comprising administering a therapeutic amount of an extract of Eleutherococcus senticosus maxim to the subject. Suitable subjects include humans. The Eleutherococcus senticosus maxim extract treats disorders mediated by both TNF-α and TNF-β.

[0014] Inhibition and mediation can occur through both prevention of cellular production of TNF and through competitive antagonism or other incapacitation of extant TNF in the subject or cell line. Since the Eleutherococcus senticosus maxim is a TNF inhibitor, the pathologies treated will be the result of TNF mediated harm. Typically, such TNF mediated harm will be the result of an overproduction of TNF. Examples of TNF mediated pathologies included toxic shock, cancer and chronic diarrhea.

[0015] Definitions and Nomenclature:

[0016] It is to be understood that this invention is not limited to specific synthetic methods, specific pharmaceutical carriers, or to particular pharmaceutical formulations or administration regimens, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0017] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” can include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes mixtures of compounds, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

[0018] For the purposes of this invention, the term “inhibiting” as it relates to levels of TNF in subjects and cell lines refers to both the prevention of production of TNF in the respective subjects or cell lines and also to prevention of the activity of any TNF which already is present in the subject or cell line. For example, inhibition of TNF can refer to the ability of CM-4 to prevent cellular production of TNF as in Example I with the HL-60 cell line. In addition, as shown in Example II below, inhibition of TNF can also refer to the ability of CM-4 to reduce the deleterious effects of existing TNF levels. Thus, in Example II, CM-4 was able to significantly reduce reverse transcriptase activity (i.e. viral replication) in OM-10.1 cells despite the presence of levels of TNF sufficient to stimulate viral replication.

[0019] By the term “TNF inhibiting amount” of a composition as provided herein is meant a nontoxic but sufficient amount of the TNF inhibiting composition to provide the desired activity. As will be pointed out below, the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Thus, it is not possible to specify an exact “TNF inhibiting amount.” However, an appropriate TNF inhibiting amount may be determined by one of ordinary skill in the art using only routine experimentation.

[0020] The Eleutherococcus senticosus maxim extract can exist in pharmaceutically acceptable carriers. A “pharmaceutically acceptable” material is one that is not biologically or otherwise undesirable, i.e., a material that may be administered to an individual along with the selected composition without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. One example of the extract in a suitable carrier is CM-4 as described below.

[0021] It must also be understood that the term ‘subjects’ as used herein is meant to include animals including humans. In addition, the dilutions of Eleutherococcus extracts are meant to be dilutions in cell culture medium or other neutral carrier. For example, a 1:300 dilution includes 1 part Eleutherococcus extract to 300 parts cell culture medium. By “extract” it is meant an active composition obtained from Eleutherococcus senticosus maxim wherein the Eleutherococcus root is a primary component either in solid, liquid, paste or the like.

[0022] Extract Preparation:

[0023] Extracts can be obtained by various methods. One extract, CM-4, is a protic extract of that Eleutherococcus root in an ethanol-water extract.

[0024] The preferred process for preparing Eleutherococcus senticosus maxim extract is as follows. Eleutherococcus senticosus maxim root is extracted, shaken free of dirt and sand, and washed with cold flowing water. The root is then dried by baking for one hour at 80° C. After baking, the roots are allowed to continue drying in a vacuum drier at 60° C. or in a warm, dry and well-ventilated location. Then roots are selected based on the following criteria: 1) the root is whole or in small parts, the bark on the outside is a yellow-brown color and internally the root is white, 2) the outside bark of the root is wrinkled, indicating that it has dried properly, 3) breaking the root open reveals a white, slightly fibrous, strongly scented aromatic characteristic odor, and 4) the taste is of the root is spicy and slightly bitter.

[0025] The selected roots are then ground into a medium-coarse powder. This powder is mixed with a protic solvent such as 40% ethanol (60% water) by volume for the extraction (1 kg powder per liter of 40% ethanol) and the mixture is heated (Pharmacopeia of USSR, page 522). The extraction can be accomplished with other protic solutions, such as water or alcohol-water solutions, for example 10-100% ethanol solutions with water.

[0026] The resulting extract is a darkish brown fluid of a specific color, is transparent, and is bitter. This extract has a dry residue of not less than 6% (Pharmacopeia, supra, p. 253), and an alcohol content of not less than 33% (Pharmacopeia, supra, p. 813).

[0027] Administration:

[0028] The Eleutherococcus senticosus maxim extract of the invention may be conveniently formulated into various pharmaceutical compositions composed of Eleutherococcus senticosus maxim extract in association with a pharmaceutically acceptable carrier. See, e.g., Remington's Pharmaceutical Sciences, latest edition, by E. W. Martin (Mack Publ. Co., Easton PA) discloses typical carriers and conventional methods of preparing pharmaceutical compositions that may be used in conjunction with the preparation of formulations.

[0029] Depending on the intended mode of administration, the pharmaceutical compositions may be in the form of solid, semi-solid or liquid dosage forms, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, lotions, creams, gels, or the like, preferably in unit dosage form suitable for single administration of a precise dosage. The compositions will include, as noted above,-an effective amount of Eleutherococcus senticosus maxim extract in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.

[0030] For solid compositions, conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc., Eleutherococcus senticosus maxim extract as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, referenced above.

[0031] For oral administration, fine powders or granules may contain diluting, dispersing, and/or surface active agents, and may be presented in water or in a syrup, in capsules or sachets in the dry state, or in a nonaqueous solution or suspension wherein suspending agents may be included, in tablets wherein binders and lubricants may be included, or in a suspension in water or a syrup. Where desirable or necessary, flavoring, preserving, suspending, thickening, or emulsifying agents may be included. Tablets and granules are preferred oral administration forms, and these may be coated.

[0032] Parenteral administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795, which is incorporated by reference herein.

[0033] In preferred embodiments, the Eleutherococcus senticosus maxim extract can be administered on a daily basis, in an amount equivalent to about 0.5-7 ml of an about 27.5% by volume ethanol in water extract of Eleutherococcus. In particularly preferred embodiments of administration to a human subject, an equivalent of about 3.3 ml of Eleutherococcus senticosus maxim extract is administered to the subject on a daily basis. The Eleutherococcus senticosus maxim extract can be taken as a single dose daily. Alternatively, and preferably, an extract is administered to the subject before breakfast daily, and an equivalent of about 1.1 ml of Eleutherococcus senticosus maxim extract is administered to the subject daily at lunch.

[0034] Experimental

[0035] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the Eleutherococcus senticosus maxim extract used in the methods claimed herein is made and evaluated, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have-been-made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for.

EXAMPLE I HL-60

[0036] HL-60 cells are a human leukemia cell line known to produce high levels of TNF in response to Phorbol myristate acetate (PMA). In order to assay the effects of Eleutherococcus senticosis maxim extract on HL-60, HL-60 cells (2×10⁶) in supplemented RPMI-1640 medium were incubated four hours in the presence of various dilutions of extract—in this case CM-4. PMA was added and the incubation continued at 37° C. in 95% air 5% CO₂. TNF production was determined by ELISA. Controls were cells alone, cells with 33% ethanol, and cells with pentoxaphyllin. The results are detailed in FIG. 1.

[0037] The results in FIG. 1 show the effect of CM-4 on TNF production in the HL-60 cell line. Specifically, HL-60 showed a marked decrease in TNF production upon treatment with 1:100 dilution CM-4 and 1:500 dilution CM-4. At the 1:100 dilution TNF production was less than 5 pg/ml whereas the control registered about 70 pg/ml. At 1:500, CM-4 limited TNF production to less than 10 pg/ml. See FIG. 1. In addition, addition of pentoxaphyllin resulted in very little TNF production (<5 pg/ml). Therefore, CM-4 in dilutions from about 1:100 to 1:500 proved to be highly effective in inhibiting TNF production in HL-60 cells.

EXAMPLE II OM 10.1

[0038] OM 10.1 is a derivative of the HL-60 cell line that is latently infected with HIV-1. In response to TNFα, HIV is induced to produce viral particles.

[0039] The OM 10.1 cell line, stock cultures, obtained from the AIDS Research and Reference Reagent Program, NIAID, were subjected to 2 passages in the presence of 5 μg/ml AZT prior to use in order to decrease background p24 and reverse transcriptase (RT) levels due to superinfection. The cells thus prepared demonstrated increased p24 and RT levels when subjected to stimulation with TNF-α with a concomitant decrease in CD4 expression as measured by FACS analysis following incubation with OK T4 antibody (Becton-Dickenson).

[0040] For experimental use, OM 10.1 cells were harvested from a stock culture and washed by centrifugation with Dulbecco's phosphate-buffered saline. The cells were resuspended in cell culture medium (RPMI 1640 medium) containing 10% fetal bovine serum (heat-inactivated at 56° C. for 30 minutes, 100 IU/ml penicillin, 100 μg/ml streptomycin, 20 μg/ml gentamicin, 8 μg/ml tylosin, and 25 mM HEPES). The cultures were maintained in a humidified atmosphere of 5% CO₂ in disposable plastic cell cultureware. The cells were seeded at 800,000 cells/2 ml/well in six well plates and an equal volume of drug dilution in cell culture medium was added. Following 1 hour of incubation, 20 U/ml of TNF-α was added to appropriate wells. Culture fluids were harvested after 48 hours of incubation for determination of RT activity and p24 content. Standard methods were used in the RT assays and in the ELISA assays for HIV p24 antigen. The results of this assay are summarized in Table 1.

[0041] Table 1 shows the effect of varying concentrations of CM-4 on HIV replication by inhibition of TNF-α in latently infected cells. The data indicate that at dilutions of from about 1:300 to 1:600, the CM-4 extract is not toxic to the subject cells. Furthermore, the data indicate that reverse transcriptase activity is significantly inhibited in OM-10.1 cells by the addition of the CM-4 extract when TNF-α is present. Therefore, the CM-4 extract was effective in preventing stimulation of virus production/replication when administered at non-cytotoxic levels. TABLE 1 Evaluation of Eleutherococcus senticosus maxim extract for Inhibition of the Stimulation by TNF-α of Active Virus Production in OM 10.1 Cells Latently Infected with HIV TNF-α CM-4^(A) CPM (RT)^(B) Viability^(C) Cytotoxicity % S^(D) % I^(E) — — 10804 — — — — — 1:200 5157 70% Toxic — — — 1:300 13324 92% Non-toxic 2 — — 1:400 12231 92% Non-toxic 1 — — 1:600 20027 96% Non-toxic 7 — + — 148547 — — — — + 1:200 9125 62% Toxic — — + 1:300 31770 87% Non-toxic — 85 + 1:400 58825 84% Non-toxic — 65 + 1:600 91779 95% Non-toxic — 42 

What is claimed is:
 1. A method of inhibiting tumor necrosis factor (TNF) in a subject comprising administering to the subject a TNF inhibiting amount of an extract from Eleutherococcus senticosus maxim.
 2. The method of claim 1, wherein the subject is a human.
 3. The method of claim 1, wherein the TNF comprises TNF-α.
 4. The method of claim 1, wherein the TNF comprises TNF-β
 5. The method of claim 1, wherein the TNF inhibiting amount is from about 1:300 dilution to about 1:600 dilution.
 6. The method of claim 1, wherein the TNF inhibiting amount is about a 1:300 dilution.
 7. The method of claim 1, wherein the extract is CM-4.
 8. A method inhibiting TNF in a cell comprising administering to the cell a TNF inhibiting amount of an extract from Eleutherococcus senticosus maxim.
 9. The method of claim 8, wherein the TNF comprises TNF-α.
 10. The method of claim 8, wherein the TNF comprises TNF-β.
 11. The method of claim 8, wherein the TNF inhibiting amount is from about 1:300 dilution to about 1:600 dilution.
 12. The method of claim 8, wherein the TNF inhibiting amount is about a 1:300 dilution.
 13. The method of claim 8, wherein the extract is CM-4.
 14. A method of treating a TNF mediated disorder in a subject, comprising administering a therapeutic amount of an extract of Eleutherococcus senticosus maxim to the subject.
 15. The method of claim 14, wherein the TNF comprises TNF-α.
 16. The method of claim 14, wherein the TNF comprises TNF-β.
 17. The method of claim 14, wherein the TNF inhibiting amount is from about 1:300 dilution to about 1:600 dilution.
 18. The method of claim 14, wherein the TNF inhibiting amount is about a 1:300 dilution.
 19. The method of claim 14, wherein the TNF mediated disorder is selected from the group consisting of toxic shock, cancer and chronic diarrhea.
 20. The method of claim 14, wherein the extract is CM-4.
 21. The method of claim 14, wherein the subject is a human. 